In liquid chromatography a liquid sample is passed by a flowing stream of liquid solvent the mobile phase) through a column packed with particulate matter (the stationary phase). While passing through the column the various components in the sample separate from one another by adsorbing and desorbing from the stationary phase at different rates such that these individual components elute from the column at different times. The separated components then flow through a detector which responds to each component both qualitatively and quantitatively, thereby providing information to the user about the constituents of the sample.
Liquid chromatography systems often use mixtures of solvents or buffers as the mobile phase and, when operating in the gradient chromatography mode, the components of the mixture are changed over time.
Gradient generation has been an integral part of chromatography since its development. The precise metering and blending of the solvents has been optimized on an analytical scale (less than 1 ml/min), but has been only marginally successful on a large scale. The two main ways to meter solvents to perform gradients are classified as "high pressure", and "low pressure" approaches. High pressure gradient generation systems perform the mixing of the solvents on the outlet (high pressure) side of the pumps. Usually, each solvent is fed through an individual pump. Proportioning is controlled by varying flow rates of each pump for the correct proportions and mixing the two streams at a splitter. Drawbacks of this technique include:
1) Cost--The requirement of two pumps, or separate drive trains on the duplex or multi-headed pumps, is expensive. High pressure gradient systems require two pumps, each equal in capacity to the single pump used in a low pressure gradient system, to deliver 0 to 100% proportioning. Also, additional hardware is usually required, such as check valves to prevent over back-pressure on the alternate head or pump. PA1 2) Performance--In generating gradients where one constituent proportion is less than 10%, efficiency of the pump decreases, causing inaccurate gradient profiles. The accuracy and precision of the proportioning is affected by motor speed and stroke length settings, especially with diaphragm and plunger-type pumps. PA1 1) Valve/Pump Synchronization--Since of the diaphragm or plunger types have a stroking speed, and the valves have a cycle time for opening and closing, synchronization of operation will appear causing wave-like proportioning. Prior art systems used complicated algorithms (U.S. Pat. No. 4,595,496) to predict possible synchronizations. PA1 2) Fine-Tuning--Because of differences in viscosity of many of the fluids used for proportioning, the valve and cycle times have to be fine-tuned for specific operations. Previous systems included a two-way valve for each solvent to be proportioned; however, accuracy is degraded when matched valves are used to proportion solvents of different viscosities. PA1 3) Valve Lag Time--The difference in actuation time from one position to the other in a valve introduces a source of inaccuracy to the proportioning by the valve. This is an inherent characteristic of all valves. PA1 4) Mixing--Better mixing is required for correct proportioning between solvents. Long cycle times required to accommodate lag time effects must be offset by large volume or high efficiency mixers. PA1 a) Lag Time--For valves using an air impulse to open, spring to close actuation, or vice versa, there is a significant amount of time required for the valve to return to the initial position. The lag time is associated with the spring returning and the release of air pressure. The "initial" position is one in which a designated valve inlet is normally open to flow from the reservoir associated therewith. PA1 b) Pump/Valve Synchronization--If a pump controlled by stroking speeds, such as a diaphragm or plunger pump, and a valve cycling by time for proportioning are used in conjunction, there are stroking speeds and cycle time multiples where synchronization can occur. Pump/valve synchronization produces inconsistent gradient profiles causing unreliable chromatographic results. Historically, U S. Pat. No. 4,595,496, determined all possible stroking speeds and cycle time multiples where synchronizations may occur and eliminated them as setpoints in their programming routines. Permutations could be in the hundreds. PA1 c) Mixing and Accuracy--Once the solvents are proportioned, the solvents have to be blended into a consistent mixture. The hold-up volume from the point of proportioning to the column inlet should be minimized to reduce the time required to obtain the desired composition when proportioning is changed. Shorter cycle times allow smaller mixing devices and/or less hold-up volume in the systems. PA1 d) Microprocessor Control--Some large scale chromatographic operations use a microprocessor controller for gradient operations and another microprocessor controller for other process control operations. Multiple microprocessors for various controls may be difficult and confusing to integrate, expensive, and not very flexible.
One advantage of high pressure gradient is that there is no risk to gradient performance due to pump and valve synchronization.
Low pressure gradient generation systems perform the mixing of the solvents at the inlet (low pressure) side of the pump. Only one pump is provided in the system to proportion two or more solvents. Valves at the inlet of the pump control the proportions of solvents. Mixing usually has been accomplished in the pump heads or at the pump outlet. Drawbacks of this technique include:
The low pressure gradient system offers a low cost alternative to the high pressure system.
Recent examples of the prior art in this field are U.S. Pat. Nos. 4,437,812 and 4,595,496. The U.S. Pat. No. 4,437,812 describes a liquid chromatography system that uses a single reciprocating pump for gradient elution of components from a plurality of reservoirs where the pumping cycle comprises a plurality of fill strokes with intervening pumping strokes, With proportioning valves being used to admit the components according to a programmed ratio from particular reservoirs into the pump chamber during a given fill stroke.
The U.S. Pat. No. 4,595,496 patent employs a dedicated microprocessor to drive both a pump and fluid switching valves, and includes means for generating a ratio between the time to connect all of the reservoirs selected for actuation and the cycle time for a pump draw stroke. Selection of an appropriate ratio controls the relative phasing between the switching valve cycle and pump draw stroke such that the beginning of each switching valve cycle occurs at a different point of the pump draw stroke, thereby providing a desired averaging of the intake nonuniformities and producing more accurate solvent mixtures.
The U.S. Pat. No. 4,437,812 patent thus discloses a system which has the disadvantages of the necessity for fine tuning the pair of two-way valves in the system, wave proportioning as the result of pump/valve synchronization and an independent controller for gradient generation, while the system of the U.S. Pat. No. 4,595,496 patent has disadvantages in that 1) a large number of valve cycle times and pump cycle times with integer ratios must bc eliminated from consideration; 2) there is a necessity for fine tuning the pair of two-way valves; and 3) an independent controller is needed for gradient generation in addition to the controller for remaining system process functions.
Thus it is apparent that while a substantial effort has been put forth to solve the problems in the field noted above, all efforts have thus far fallen short of providing an accurate, reproducible technique for metering liquids in controlled proportions with minimum cost and complexity in this field of large scale (100 ml/min or more) liquid chromatography.